Bcl2L12調控細胞凋亡，但它在不同癌症的角色一直有著爭議。Bcl2L12在惡性腦瘤(GBM)透過與p53、αB-crystallin、caspase-3以及caspase-7參與後粒線體細胞凋亡的調控。Bcl2L12 被認為會與Bcl-xL交互作用，此外也帶有一個BH3-相似區段。因此，我們調查此一BH3-相似區段與Bcl2L12的抗細胞凋亡特性是否有關；並且ABT-737是否可以與TMZ併用。發現胺基酸192-220 的區域對Bcl2L12-Bcl-xL 之間的交互作用相當重要。在U87MG細胞過表現h1或h2突變蛋白較之野生型蛋白，會使細胞凋亡標記蛋白再活化且也會造細胞色素c釋放。併用ABT-737與TMZ會刺激出更嚴重的細胞凋亡反應。因此，證實的確有一個具功能性的BH3-相似區段，它與抗細胞凋亡特性以及TMZ誘發的細胞自噬作用有關。相對地，Bcl2L12在乳癌是一個良好的預後因子，但背後的因素不明朗。所以我們調查Bcl2L12跟它的亞型分子mRNA表現是否與乳癌的惡化或特定的分類有關。在106不同乳癌檢體中，兩個mRNA分子表現在不同的分期、病理分期的乳癌之間並無差異。 然而，高Bcl2L12 mRNA表現與高病理分期以及三負性乳癌有關。並且，Bcl2L12與它的亞型分子在非三負性乳癌中與淋巴轉移的嚴重程度有關。之後，應用miRanda檢索網站選出可能與Bcl2L12作用的微小RNAs，偵測pri-miR-182 以及pri-miR-1271在乳癌細胞株以及50個檢體的表現。結果pri-miR-182和pri-miR-1271都可以在T47D 以及部份的檢體偵測到。選殖微小RNA的前驅物並與不同的3’UTR片段以及微小RNA結合位突變螢光載體一同轉染到不同的乳癌細胞株。Dual-Glo試驗顯示兩個微小RNAs可能透過結合到3’UTR，都會調降螢光基因的表現；再者，miR-182較之miR-1271對Bcl2L12有較強的調控作用。

Bcl2L12 regulates apoptosis, but the different roles of Bcl2L12 in different cancer types remains controversial. In glioma multiforme (GBM), Bcl2L12 is involved in post-mitochondrial apoptosis regulation via the interrelationships between p53, αB-crystallin, and caspase-3 and -7. Bcl2L12 is suspected to interact with Bcl-xL and harbors a BH3-like domain. We investigated whether this BH3-like domain correlates to Bcl2L12’s anti-apoptotic properties, and the implications for the possible combination use of ABT-737 and TMZ in GBM. We found that the region encompassing residues 192-220 is crucial for Bcl2L12-Bcl-xL interaction. Overexpression of either the h1 or h2 mutant in U87MG resulted in reactivation of apoptotic markers and caused cytochrome c release, compared to wild-type protein. Combining ABT-737 with TMZ induced a superior apoptotic event. Thus, a functional BH3-like domain in Bcl2L12 is linked to both its anti-apoptotic properties and TMZ-induced autophagy. In contrast, in breast cancer (BCa) Bcl2L12 is a good prognostic marker, but its mechanistic role is unclear. We therefore examined whether mRNA expression of Bcl2L12 and its variant is associated with BCa progression or a specific BCa subtype. Expression of the two mRNA markers was not significantly different across the different stage, grade and TNM classifications in 106 paraffin-embedded breast cancer specimens of different stages. However, high Bcl2L12 mRNA expression was associated with the high-grade BCa and TNBCs. In addition, the interplay between Bcl2L12 and its variant may be associated with high lymph node metastasis in non-TNBC tumors. We used the miRanda algorithm to investigate Bcl2L12-interacting miRNAs, and measured pri-miR-182 and pri-miR-1271 across different BCa lines in 50 clinical specimens. Both pri-miR-182 and pri-miR-1271 were detected in T47D cells and a proportion of tumor specimens. The precursors of both these miRNAs were cloned and co-transfected with different 3’UTR fragments and miRNA binding mutants into BCa lines. Dual-Glo assay showed that both miRNAs downregulated luciferase expression, which may bind to 3’UTR. In addition, miR-182 was more dominant in regulating Bcl2L12 than miR-1271.

Table of Contents
誌謝 ii
中文摘要 iii
Abstract iv
List of Figures圖次 ix
List of Tables 表次 xi
Terminology: abbreviations and symbols xii
Chapter 1 1
Research Part I. Role of Bcl2L12 with BH3-like domain in regulating apoptosis and TMZ-induced autophagy: The prospective combination of ABT-737 and TMZ for treating glioma 1
1.1 Summary 1
1.2 Epidemiology of glioblastoma 2
1.3 Etiology of glioblastoma 2
1.4 Clinical treatment of glioma 3
1.5 Bcl2 family protein and apoptosis 5
1.6 Bcl2L12 and cancer 8
1.7 Rationale and objectives 10
1.8 Materials and Methods 11
1.8.1 Cloning 11
1.8.2 Yeast two-hybrid assay 12
1.8.3 Site-directed mutagenesis 13
1.8.4 Protein structure prediction 13
1.8.5 Cell culture, transfection and treatments 13
1.8.6 Quantitative PCR 14
1.8.7 Western blotting 15
1.8.8 Statistical analysis 15
1.9 Results of Research Part I 16
1.9.1 Bcl2L12192-220 is the region responsible for interaction with Bcl-xL 16
1.9.2 The BH3-like domain of Bcl2L12 is located at residue 211-226 19
1.9.3 Hydrophobic residue h1 (L213), h2(L217) and h4 (I224) within the Bcl2L12 BH3-like domain are crucial for Bcl2/Bcl-xL interaction in the yeast two hybrid system 22
1.9.4 Ectopically expressed Bcl2L12 BH3-like domain mutant causes reactivation of apoptotic markers 23
1.9.5 In combination of TMZ and ABT-737 exerts a better apoptotic induction than each used alone in U8MG cell 27
1.10. Discussion of Research Part I 29
Chapter 2 37
Research Part II. Effect of inhibiting Bcl2L12 expression on autophagy via the BH3-like domain in glioma cells- another role of Bcl2L12 in TMZ-induced autophagy 37
2.1. Summary 37
2.2. Introduction of Research Part II 38
2.2.1. A new proposed mechanism for Bcl2L12 as anti-apoptotic Bcl2 protein in glioma 38
2.2.2. Cancer therapy and drug-induced autophagy 38
2.3. Rationale and objectives 40
2.4. Materials and Methods 41
2.4.1. Cloning 41
2.4.2. Yeast two-hybrid assay 42
2.4.3. Site-directed mutagenesis 42
2.4.4. Protein structure prediction 43
2.4.5. Cell culture, transfection and treatments 43
2.4.6. Western blotting 44
2.4.7. Statistical analysis 44
2.5. Results of Research Part II 45
2.5.1. Bcl2L12 retains a BH3-like domain on its α-9 helix and this 12-residue motif is conserved among the Bcl2 family proteins 45
2.5.2. Bcl2L12 did not interact with Beclin-1, but shared similar binding partnerships to Beclin-1 in interaction with Bcl2 and Bcl-xL, but not Mcl-1 46
2.5.3. BH3-like domain L213A mutant restores apoptotic markers activities in U87MG, but not T98G cells 49
2.5.4. Combination use of TMZ and ABT-737 triggered higher apoptosis than either alone in U87MG cells 52
2.5.5. TMZ triggers an autophagy-apoptosis shift event in a time-dependent manner in U87MG and H4, but not in T98G cells 53
2.5.6. GSK3-mediated phosphorylation on Ser156 and BH3-like domain both contribute to the anti-apoptotic property of Bcl2L12 and drug-induced autophagy in U87MG and H4 cells, but not in T98G cells 55
2.5.7. Blocking TMZ-induced autophagy lead to a strong activation of apoptotic markers as well as TP53 expression in U87MG cells. 59
2.6. Discussion of Research Part II 60
Chapter 3 68
Research Part III. Differential roles of Bcl2L12 and its short variant in breast cancer lymph node metastasis 68
3.1. Summary 68
3.2. Breast Cancer 69
3.3. Bcl2L12 and its variant in cancer biology 69
3.4. Rationale and objectives 71
3.5. Materials and Methods 71
3.5.1. Tissue collection 71
3.5.2. RNA extraction 74
3.5.3. cDNA synthesis 74
3.5.4. Quantitative PCR 74
3.5.5. Statistical analysis 78
3.6. Results of Research Part III 78
3.7. Discussion of Research Part III 86
Chapter 4 91
Research Part IV. Investigation of miRNA candidates that possibly regulate the expression of Bcl2L12 in breast cancer 91
4.1. miRNA and cancer 91
4.2. Bcl2L12-interacting miRNA 94
4.3. Rational and Objectives 96
4.4. Materials and Methods 97
4.4.1. Cell culture and tissue collection 97
4.4.2. Target miRNA predictions 97
4.4.3. RNA isolation and cDNA thesis 98
4.4.4. Vector construction 98
4.4.5. Electroporation 99
4.4.6. Luciferase assays 99
4.5. Results of Research Part IV 100
4.6. Discussion of Research Part IV 112
Conclusions 115
References 117
Appendix: Publications Arising from this dissertation 130

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